LKB1基因对卵巢颗粒细胞类固醇激素生成相关基因的调控作用

LKB1 Regulates Steroids Synthesis Related Genes Expression in Bovine Granulosa Cells

【背景】颗粒细胞类固醇激素的合成能力对卵泡发育及成熟具有重要作用,但其关键的调控因子尚不完全清楚。笔者前期的研究表明肝激酶B1(liver kinase B1,LKB1)基因参与细胞的脂类代谢,类固醇激素的合成与脂类代谢密切相关,并且有研究结果亦显示LKB1敲除可引起小鼠卵巢早衰,表明LKB1对维持卵巢的功能很关键,其在颗粒细胞的确切功能需要进一步研究。【目的】探究LKB1在牛卵泡中的表达模式及其对颗粒细胞类固醇激素生成相关基因的调控作用,为母牛繁殖生理调控研究提供理论依据。【方法】采用免疫组织化学染色对LKB1蛋白在卵泡中进行定位研究;同时分离培养牛原代颗粒细胞,并以促卵泡素受体(follicle stimulating hormone receptor, FSHR)蛋白作为标记基因,细胞免疫荧光染色鉴定颗粒细胞及纯度;然后以原代颗粒细胞为模型,采用siRNA沉默LKB1的技术,利用qRT-PCR方法检测LKB1功能缺失对类固醇激素合成相关基因表达的影响,另一方面采用腺病毒过表达LKB1,qRT-PCR和ELISA技术验证LKB1对类固醇激素合成相关基因表达的调控作用及雌二醇分泌。【结果】1) LKB1蛋白在卵泡中的细胞均表达,但颗粒细胞的染色信号强于膜细胞,进一步的定量分析显示颗粒细胞的表达量显著高于卵泡膜细胞。2)分离培养的牛原代卵泡颗粒细胞贴壁生长、细胞形态多呈圆形,能被颗粒细胞标志基因FSHR抗体标记。3) RNAi技术能显著抑制LKB1的表达。与对照相比,siRNA1和siRNA2干扰LKB1的效率分别为48%(P<0.05)和52%(P<0.05);沉默LKB1显著降低颗粒细胞类固醇激素合成基因STAR (P<0.01)、CYP11A1 (P<0.01)和CYP19A1 (P<0.05)的表达,分别下调了约为对照组的60%、80%和50%。4) LKB1过表达腺病毒及对照组对颗粒细胞均具有高的感染效率,LKB1过表达效率高达10倍(P<0.01);过表达LKB1显著上调STAR (P<0.01)、CYP11A1(P<0.01)和CYP19A1 (P<0.05)的表达,进一步研究显示LKB1基因功能获得促进颗粒细胞雌二醇的分泌(P<0.05)。【结论】LKB1在卵泡颗粒细胞中高表达,促进类固醇激素生成基因STAR、CYP11A1和CYP19A1的表达和雌二醇的分泌。本研究将为LKB1调控牛颗粒细胞类固醇激素合成的功能提供直接的理论依据。

【Background】 The Steroids synthesis capacity of ovarian granulosa cells plays the important roles in the development and maturation of follicles, however, the key regulators were involved in this process remains largely unknown. Our previously research reported that Liver kinase B1(LKB1) influenced the cellular lipid metabolism, which is close associated with steroids synthesis. Further, another study showed that knockout of LKB1 caused premature ovarian failure in mice. 【Objective】 The aim of this study was to study the expression pattern of LKB1 in bovine follicle and its regulation on steroid synthesis related genes expression in granulosa cells(GCs),and provided a theoretical basis for the research of the reproductive physiological regulation in the cow.【Method】The expression pattern of LKB1 in follicle was detected by immunohistochemically assay. Then the primary follicular granulosa cells were isolated and identified by immunofluorescence staining incubated by follicle stimulating hormone receptor(FSHR) antibody. Next, these verified granulosa cells were used as the cell model. On one hand, LKB1 loss-of-function was mediated by siRNAs. qRT-PCR was performed to measure LKB1 regulation of steroid hormone synthesis related genes expression.On the other hand, LKB1 gain-of-function was mediated by adenovirus. qRT-PCR and ELISA analysis were carried out to confirm the changes of above detected genes influenced by LKB1 and estradiol(E2) secretion, respectively. 【Result】 The data showed that:1) LKB1 protein expressed in all cell types of follicles and the positive signal in granulosa cells is significantly higher than that of theca cells, which is verified by quantitative analysis. 2) The morphology of isolated bovine follicular granulosa cells was shape of round, which were specifically labeled by follicle stimulating hormone receptor(FSHR) using immunofluorescence staining, with 95% of positive cells. 3) The interference efficiency of LKB1 treated by siRNA1 and siRNA2 was respectively 48%(P<0.05) and 52%(P<0.05) to that of control. Knockdown of LKB1 significantly down-regulated mRNA levels of STAR(P<0.01), CYP11A1(P<0.01) and CYP19A1(P<0.05), with the 60%, 80% and 50% decrease to those of the control. 4) The highly infected efficiency was observed infected by LKB1-OE and control adenovirus. In contrast, overexpression of LKB1 dramatically increased mRNA levels of STAR(P<0.01), CYP11A1(P<0.01) and CYP19A1(P<0.05), which was associated with elevation of E2 secretion. 【Conclusion】In summary, LKB1 was highly expressed in follicular granulosa cells, which promoted the expression of steroids synthesis related genes and E2 secretion. This result provides directly theoretical evidence for the LKB1 regulation of steroids hormone synthesis in bovine.